Publications by authors named "Nir Hacohen"

186 Publications

A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies.

Nat Commun 2021 Sep 17;12(1):5506. Epub 2021 Sep 17.

Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 10 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.
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http://dx.doi.org/10.1038/s41467-021-25777-zDOI Listing
September 2021

Spatially organized multicellular immune hubs in human colorectal cancer.

Cell 2021 Sep 26;184(18):4734-4752.e20. Epub 2021 Aug 26.

Department of Immunology, HMS, Boston, MA, USA.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.
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http://dx.doi.org/10.1016/j.cell.2021.08.003DOI Listing
September 2021

Viral Load Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2 in Hospitalized Individuals With Coronavirus Disease 2019.

Open Forum Infect Dis 2021 Aug 17;8(8):ofab153. Epub 2021 Apr 17.

Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kinetics remain understudied, including the impact of remdesivir. In hospitalized individuals, peak sputum viral load occurred in week 2 of symptoms, whereas viremia peaked within 1 week of symptom-onset, suggesting early systemic seeding of SARS-CoV-2. Remdesivir treatment was associated with faster viral decay.
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http://dx.doi.org/10.1093/ofid/ofab153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083268PMC
August 2021

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

Mol Cell Proteomics 2021 Aug 12;20:100133. Epub 2021 Aug 12.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address:

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.
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http://dx.doi.org/10.1016/j.mcpro.2021.100133DOI Listing
August 2021

Differentiation of exhausted CD8 T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory.

Nat Immunol 2021 08 26;22(8):1030-1041. Epub 2021 Jul 26.

Division of Gastroenterology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8 T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.
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http://dx.doi.org/10.1038/s41590-021-00982-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8323980PMC
August 2021

Phenotype, specificity and avidity of antitumour CD8 T cells in melanoma.

Nature 2021 08 21;596(7870):119-125. Epub 2021 Jul 21.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8 T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.
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http://dx.doi.org/10.1038/s41586-021-03704-yDOI Listing
August 2021

Vaccine serologic responses among transplant patients associate with COVID-19 infection and T peripheral helper cells.

medRxiv 2021 Jul 14. Epub 2021 Jul 14.

Background: Therapeutically immunosuppressed transplant recipients exhibit attenuated responses to COVID-19 vaccines. To better understand the immune alterations that determined poor vaccine response, we correlated quantities of circulating T and B cell subsets at baseline with longitudinal serologic responses to SARS-CoV-2 mRNA vaccination in heart and lung transplant recipients.

Methods: Samples at baseline and at approximately 8 and 30 days after each vaccine dose for 22 heart and lung transplant recipients with no history of COVID-19, four heart and lung transplant recipients with prior COVID-19 infection, and 12 healthy controls undergoing vaccination were analyzed. Anti-spike protein receptor binding domain (RBD) IgG and pseudovirus neutralization activity were measured. Proportions of B and T cell subsets at baseline were comprehensively quantitated.

Results: At 8-30 days post vaccination, healthy controls displayed robust anti-RBD IgG responses, whereas heart and lung transplant recipients showed minimally increased responses. A parallel absence of activity was observed in pseudovirus neutralization. In contrast, three of four (75%) transplant recipients with prior COVID-19 infection displayed robust responses at levels comparable to controls. Baseline levels of activated PD-1 HLA-DR CXCR5 CD4 T cells (also known as T peripheral helper [T ] cells) and CD4+ T cells strongly predicted the ability to mount a response.

Conclusions: Immunosuppressed patients have defective vaccine responses but can be induced to generate neutralizing antibodies after SARS-CoV-2 infection. Strong correlations of vaccine responsiveness with baseline T and CD4 T cell numbers highlights a role for T helper activity in B cell differentiation into antibody secreting cells during vaccine response.
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http://dx.doi.org/10.1101/2021.07.11.21260338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288159PMC
July 2021

Plasma P-selectin is an early marker of thromboembolism in COVID-19.

medRxiv 2021 Jul 14. Epub 2021 Jul 14.

Coagulopathy and thromboembolism are known complications of SARS-CoV-2 infection. The mechanisms of COVID-19-associated hematologic complications involve endothelial cell and platelet dysfunction and have been intensively studied. We leveraged a prospectively collected acute COVID-19 biorepository to study the association of plasma levels of a comprehensive list of coagulation proteins with the occurrence of venous thromboembolic events (VTE). We included in our analysis 305 subjects with confirmed SARS-CoV-2 infection who presented to an urban Emergency Department with acute respiratory distress during the first COVID-19 surge in 2020; 13 (4.2%) were subsequently diagnosed with venous thromboembolism during hospitalization. Serial samples were obtained and assays were performed on two highly-multiplexed proteomic platforms. Nine coagulation proteins were differentially expressed in patients with thromboembolic events. P-selectin, a cell adhesion molecule on the surface of activated endothelial cells, displayed the strongest association with the diagnosis of VTE, independent of disease severity (p=0.0025). This supports the importance of endothelial activation in the mechanistic pathway of venous thromboembolism in COVID-19. P-selectin together with D-dimer upon hospital presentation provided better discriminative ability for VTE diagnosis than D-dimer alone.
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http://dx.doi.org/10.1101/2021.07.10.21260293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288158PMC
July 2021

SARS-CoV-2 viremia is associated with distinct proteomic pathways and predicts COVID-19 outcomes.

J Clin Invest 2021 07;131(13)

Brigham and Women's Hospital and.

BACKGROUNDSARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19 in small-scale cohort studies. The mechanisms behind this association remain elusive.METHODSWe evaluated the relationship between SARS-CoV-2 viremia, disease outcome, and inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using a quantitative reverse transcription PCR-based platform. Proteomic data were generated with Proximity Extension Assay using the Olink platform.RESULTSThis study included 300 participants with nucleic acid test-confirmed COVID-19. Plasma SARS-CoV-2 viremia levels at the time of presentation predicted adverse disease outcomes, with an adjusted OR of 10.6 (95% CI 4.4-25.5, P < 0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and 3.9 (95% CI 1.5-10.1, P = 0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, and endothelium/vasculature, and alterations in coagulation pathways.CONCLUSIONThese results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.FUNDINGMark and Lisa Schwartz; the National Institutes of Health (U19AI082630); the American Lung Association; the Executive Committee on Research at Massachusetts General Hospital; the Chan Zuckerberg Initiative; Arthur, Sandra, and Sarah Irving for the David P. Ryan, MD, Endowed Chair in Cancer Research; an EMBO Long-Term Fellowship (ALTF 486-2018); a Cancer Research Institute/Bristol Myers Squibb Fellowship (CRI2993); the Harvard Catalyst/Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH awards UL1TR001102 and UL1TR002541-01); and by the Harvard University Center for AIDS Research (National Institute of Allergy and Infectious Diseases, 5P30AI060354).
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http://dx.doi.org/10.1172/JCI148635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245177PMC
July 2021

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

Cell 2021 07 3;184(15):3962-3980.e17. Epub 2021 Jun 3.

Harvard Medical School, Boston, MA 02115, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, USA; Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA; Massachusetts Consortium on Pathogen Readiness, Boston, MA, USA.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.
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http://dx.doi.org/10.1016/j.cell.2021.05.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173604PMC
July 2021

Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro.

Sci Transl Med 2021 06 8;13(598). Epub 2021 Jun 8.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14 monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14 MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection.
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http://dx.doi.org/10.1126/scitranslmed.abe9599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432955PMC
June 2021

Early cross-coronavirus reactive signatures of protective humoral immunity against COVID-19.

bioRxiv 2021 May 12. Epub 2021 May 12.

The introduction of vaccines has inspired new hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against COVID-19, thus we profiled the earliest humoral signatures in a large cohort of severe and asymptomatic COVID-19 individuals. While a SARS-CoV-2-specific immune response evolved rapidly in survivors of COVID-19, non-survivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibody evolution. Given the conservation of S2 across β-coronaviruses, we found the early development of SARS-CoV-2-specific immunity occurred in tandem with pre-existing common β-coronavirus OC43 humoral immunity in survivors, which was selectively also expanded in individuals that develop paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.
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http://dx.doi.org/10.1101/2021.05.11.443609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132219PMC
May 2021

Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures, tissue-specific cell death, and cell-cell interactions.

Cell Rep Med 2021 May 3;2(5):100287. Epub 2021 May 3.

Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA.

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.
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http://dx.doi.org/10.1016/j.xcrm.2021.100287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091031PMC
May 2021

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

Nature 2021 07 29;595(7865):107-113. Epub 2021 Apr 29.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63 intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.
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http://dx.doi.org/10.1038/s41586-021-03570-8DOI Listing
July 2021

Genome-wide enhancer maps link risk variants to disease genes.

Nature 2021 05 7;593(7858):238-243. Epub 2021 Apr 7.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease. Many of the underlying causal variants may affect enhancers, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.
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http://dx.doi.org/10.1038/s41586-021-03446-xDOI Listing
May 2021

Alternative Splicing of the Inhibitory Immune Checkpoint Receptor SLAMF6 Generates a Dominant Positive Form, Boosting T-cell Effector Functions.

Cancer Immunol Res 2021 06 24;9(6):637-650. Epub 2021 Mar 24.

Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem, Israel.

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor-exhausted T cells. Here we show that in humans, SLAMF6 has three splice isoforms involving its V-domain. Although the canonical receptor inhibited T-cell activation through SAP recruitment, the short isoform SLAMF6 had a strong agonistic effect. The costimulatory action depended on protein phosphatase SHP1 and led to a cytotoxic molecular profile mediated by the expression of TBX21 and RUNX3. Patients treated with immune checkpoint blockade showed a shift toward SLAMF6 in peripheral blood T cells. We developed splice-switching antisense oligonucleotides (ASO) designed to target the relevant SLAMF6 splice junction. Our ASOs enhanced SLAMF6 expression in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The yin-yang relationship of SLAMF6 splice isoforms may represent a balancing mechanism that could be exploited to improve cancer immunotherapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0800DOI Listing
June 2021

SARS-CoV-2 Viremia is Associated with Distinct Proteomic Pathways and Predicts COVID-19 Outcomes.

medRxiv 2021 Feb 26. Epub 2021 Feb 26.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) plasma viremia has been associated with severe disease and death in coronavirus disease 2019 (COVID-19) in small-scale cohort studies. The mechanisms behind this association remain elusive.

Methods: We evaluated the relationship between SARS-CoV-2 viremia, disease outcome, inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using qRT-PCR based platform. Proteomic data were generated with Proximity Extension Assay (PEA) using the Olink platform.

Results: Three hundred participants with nucleic acid test-confirmed COVID-19 were included in this study. Levels of plasma SARS-CoV-2 viremia at the time of presentation predicted adverse disease outcomes, with an adjusted odds ratio (aOR) of 10.6 (95% confidence interval [CI] 4.4, 25.5, P<0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and aOR of 3.9 (95%CI 1.5, 10.1, P=0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, endothelium/vasculature and alterations in coagulation pathways.

Conclusions: These results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.
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http://dx.doi.org/10.1101/2021.02.24.21252357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924277PMC
February 2021

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2.

bioRxiv 2021 Feb 25. Epub 2021 Feb 25.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63 intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.
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http://dx.doi.org/10.1101/2021.02.25.430130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924267PMC
February 2021

Rationale and design of the Kidney Precision Medicine Project.

Kidney Int 2021 03;99(3):498-510

Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Renal Section, Veterans Administration Pittsburgh Healthcare System, Pittsburgh, Pennsylvania, USA.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are common, heterogeneous, and morbid diseases. Mechanistic characterization of CKD and AKI in patients may facilitate a precision-medicine approach to prevention, diagnosis, and treatment. The Kidney Precision Medicine Project aims to ethically and safely obtain kidney biopsies from participants with CKD or AKI, create a reference kidney atlas, and characterize disease subgroups to stratify patients based on molecular features of disease, clinical characteristics, and associated outcomes. An additional aim is to identify critical cells, pathways, and targets for novel therapies and preventive strategies. This project is a multicenter prospective cohort study of adults with CKD or AKI who undergo a protocol kidney biopsy for research purposes. This investigation focuses on kidney diseases that are most prevalent and therefore substantially burden the public health, including CKD attributed to diabetes or hypertension and AKI attributed to ischemic and toxic injuries. Reference kidney tissues (for example, living-donor kidney biopsies) will also be evaluated. Traditional and digital pathology will be combined with transcriptomic, proteomic, and metabolomic analysis of the kidney tissue as well as deep clinical phenotyping for supervised and unsupervised subgroup analysis and systems biology analysis. Participants will be followed prospectively for 10 years to ascertain clinical outcomes. Cell types, locations, and functions will be characterized in health and disease in an open, searchable, online kidney tissue atlas. All data from the Kidney Precision Medicine Project will be made readily available for broad use by scientists, clinicians, and patients.
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http://dx.doi.org/10.1016/j.kint.2020.08.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330551PMC
March 2021

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Nat Metab 2021 02 22;3(2):182-195. Epub 2021 Feb 22.

The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.
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http://dx.doi.org/10.1038/s42255-021-00350-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954080PMC
February 2021

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses.

Sci Transl Med 2021 02;13(581)

Experimental Cancer Genetics, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8 T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.
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http://dx.doi.org/10.1126/scitranslmed.abd8636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130008PMC
February 2021

Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma.

Nat Med 2021 03 21;27(3):515-525. Epub 2021 Jan 21.

Department of Data Sciences, Dana-Farber Cancer Institute, Boston, MA, USA.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.
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http://dx.doi.org/10.1038/s41591-020-01206-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8273876PMC
March 2021

Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection.

Nat Commun 2020 12 9;11(1):6319. Epub 2020 Dec 9.

Department of Pathology, Brigham and Woman's Hospital, Boston, MA, 02115, USA.

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41467-020-20139-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725958PMC
December 2020

Personal Neoantigen Cancer Vaccines: A Road Not Fully Paved.

Cancer Immunol Res 2020 12;8(12):1465-1469

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Personal neoantigen-based cancer vaccines are designed to target antigens arising from tumor-specific mutations within individual cancers and present a tremendous opportunity to capitalize on their favorable and intrinsic properties of escape from central tolerance and exquisite tumor specificity. With the endpoint of creating an optimal T-cell army to attack a tumor, neoantigen-based vaccines have demonstrated the ability to coax naïve T-cell recruits against epitopes that do not induce spontaneous immunity to raise long-lasting T-cell responses against multiple tumor-specific epitopes and subsequently to extend the breadth of responses, as immunity begets immunity via epitope spreading. Importantly, on both preclinical and clinical fronts, the association of T-cell responses to neoantigens and favorable outcomes has been demonstrated time and time again. We recognize, however, that the path forward remains long and winding and requires the field to address several key challenges, particularly overcoming evolved tumor escape mechanisms and optimizing vaccine-induced immunity. Some challenges stem from gaps in science that enable prediction of antigen presentation and recognition by T-cell receptors, whereas others stem from the logistical obstacles and cost of personalization. Nevertheless, with perseverance and innovative solutions, we have little doubt that the ability of neoantigen vaccination to induce potent cancer-specific T cells will fundamentally succeed in enabling greater effectiveness of a broad array of immunotherapies. We provide our perspective on the progress and the remaining challenges to realizing the opportunity of personal neoantigen cancer vaccines.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717540PMC
December 2020

The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis.

Cancer Discov 2021 Mar 17;11(3):678-695. Epub 2020 Nov 17.

Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of -mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities..
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http://dx.doi.org/10.1158/2159-8290.CD-19-1500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933049PMC
March 2021

Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients.

bioRxiv 2020 Nov 3. Epub 2020 Nov 3.

COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.
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http://dx.doi.org/10.1101/2020.11.02.365536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654866PMC
November 2020

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies.

bioRxiv 2020 Oct 30. Epub 2020 Oct 30.

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities and may serve as the basis for automated and highly parallel antibody generation.
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http://dx.doi.org/10.1101/2020.10.29.361287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605568PMC
October 2020

Key Parameters of Tumor Epitope Immunogenicity Revealed Through a Consortium Approach Improve Neoantigen Prediction.

Cell 2020 10 9;183(3):818-834.e13. Epub 2020 Oct 9.

Division of Hematology and Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Department of Medicine, University of California, San Diego, La Jolla, CA, USA.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.
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http://dx.doi.org/10.1016/j.cell.2020.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652061PMC
October 2020

SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs.

bioRxiv 2020 Oct 26. Epub 2020 Oct 26.

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.
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http://dx.doi.org/10.1101/2020.10.02.324145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536868PMC
October 2020
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